2600226013, September 7, 2007 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Association of Specific Proteolytic Processing of Bone Sialoprotein and Bone Acidic Glycoprotein-75 with Mineralization within Biomineralization Foci*
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Received for publication, February 15, 2007, and in revised form, June 19, 2007 Published, JBC Papers in Press, July 5, 2007, DOI 10.1074/jbc.M701332200
Nichole T. Huffman, J. Andrew Keightley, Cui Chaoying, Ronald J. Midura, Dinah Lovitch, Patricia A. Veno, Sarah L. Dallas, and Jeff P. Gorski1 From the Bone Biology Program, Department of Oral Biology, School of Dentistry, University of Missouri, Kansas City, Missouri 64108, Biological Mass Spectrometry and Proteomics Facility, Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110, Tibet University Medical College, Lhasa 850002, Tibet, China, and the Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195
Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 1525-m extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (4550 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblastderived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.
* This work was supported by National Institutes of Health Grants R21 DE14619
(to J. P. G.) and R01 AR052775 (to J. P. G.) and small grants from the Womens Council of the University of Missouri, Kansas City (to N. T. H.). Parts of this research were presented in preliminary form at the annual meeting of the American Society for Biochemistry and Molecular Biology, June 1216, 2004, Boston, MA; the American Society for Bone and Mineral Research, September 2327, 2005, Nashville, TN; and the American Society for Bone and Mineral Research, September 1519, 2006, Philadelphia, PA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1S3. 1 To whom correspondence should be addressed: Bone Biology Program, Oral Biology, School of Dentistry, 625 East 25th St., University of Missouri, Kansas City, MO 64108. Tel.: 816-235-2537; Fax: 816-235-5524; E-mail: Gorskij@umkc.edu.
Bone is a vascularized tissue that uniquely becomes mineralized as part of its developmental program (1). Mineralized bone serves essential vertebrate functions, including structural support, reversible storage for calcium and phosphorus, and as a reservoir for toxic metals and carbonate (2). Bone tissue is composed of osteoid; osteoblasts, which produce and mineralize new bone; osteoclasts, which resorb bone; and osteocytes, mature osteoblasts that maintain bone viability (13). Osteoid is a type I collagen-rich extracellular matrix enriched in acidic noncollagenous proteins (4). Using fetal rat calvaria cell cultures, Bellows et al. (5) showed that osteoid is unmineralized when initially deposited, and mineral crystals form within nodular structures over the following 48 72 h. Bone matrices can be classified as lamellar, based on a highly organized layered structure, or woven bone. Woven bone is formed during embryonic development, fracture healing, and at sites receiving mechanical stimulation in excess of 3,000 microstrain (6); lamellar bone replaces woven bone later in development. The question of whether bone mineralization is under direct osteoblastic control or whether it is purely a passive chemical process is under active investigation. Schinke et al. (7) have proposed that calcification reactions in vivo are passive physiochemical processes occurring readily where local mineralization inhibitors are overwhelmed. In support of this hypothesis, Murshed et al. (8) produced a calcified dermal layer in transgenic mice expressing alkaline phosphatase in skin under the control of the type I collagen chain promoter (2). Similarly, Luo et al. (9) and Murshed et al. (10) showed that matrix GLA protein is a passive local inhibitor of vascular calcification because deficient mice calcify their thoracic aorta. The latter approach emphasizes the formation of hydroxyapatite crystals as the primary experimental outcome. A second view focuses on the active role of local extracellular nucleation complexes such as biomineralization foci (11, 12), crystal ghosts (13, 14), matrix vesicles (15), and the hole regions of collagen fibrils (16) with matrix vesicles (17, 18) or with extracellular matrix phosphoproteins (12, 19, 20). We have proposed that mineralization can be divided
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EXPERIMENTAL PROCEDURES
Materials Antibodies were from several sources as follows: nonimmune rabbit IgG (EMD Biosciences), anti-BAG-75 (number 503) (anti-peptide antibody) rabbit serum (24); anti-BAG-75 (number 504) (anti-protein antibody) rabbit serum (24); antibone sialoprotein LF-100 antiserum (Larry Fisher, NIDCR, National Institutes of Health); and monoclonal anti-BSP (WV1D1(9C5)) antibody (NIH Developmental Studies Hybridoma Bank, University of Iowa). Methods Cell CultureUMR 106-01 BSP cells were passaged and cultured at 37 C and 5% carbon dioxide as described previously (12, 22) and updated briefly here. Cells were seeded at a density of 1.0 105 cells/cm2 in Growth Medium (Eagles MEM supplemented with Earles salts, 1% nonessential amino acids (Sigma), 10 mM HEPES (pH 7.2), and 10% fetal bovine serum (Hyclone)). After 24 h, the medium was exchanged with Growth Medium containing 0.5% BSA (catalog number A-1933, Sigma) instead of FBS. Sixty four hours after plating, the culture medium was exchanged with Mineralization Media (Growth Medium containing either 0.1% BSA or 10% fetal bovine serum and 7 mM BGP). Cultures were then incubated for an additional 24 h, at the end of which (88 h) the cells were either subjected to MTT assay or fixed in 70% ethanol and then extracted for protein. In some experiments, protease inhibitors, including serine protease inhibitor AEBSF [(4-(2-aminoethyl)benzenesulfonylfluoride HCl)] (EMD Biosciences), were added to cultures at 64 h after plating in Mineralization Media. Alternatively, AEBSF was added at 44 h after plating; inhibitor was then removed and exchanged for Mineralization Media at 64 h and the amount of mineralization analyzed at 88 h. Primary mouse osteoblasts were isolated from calvaria of 57-day-old mice using a modification of the method described previously (25, 26). Briefly, the calvaria were aseptically harvested, and four sequential 20-min digests were performed in 0.05% trypsin, 0.2% collagenase in Hanks balanced salt solution. Fractions 2 4 were pooled, centrifuged, and resuspended in -MEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 30 g/ml gentamicin (-Growth Medium). 2 106 cells were plated per T-75-cm2 flask and allowed to reach confluency (3 4 days). Confluent flasks were then trypsinized and plated into 12- or 24-well culture dishes for experiments at a density of 20,000 cells per cm2 growth area using media and supplements as described above. At confluency, the media were changed to -MEM containing 5% FBS, 50 g/ml ascorbic acid, 5 mM BGP, and other supplements as described above. BGP was omitted from some wells that served as an un-mineralized control. To test the effect of AEBSF, identical duplicate cultures were treated on days 3, 6, or 9 with 0.003 to 0.1 mM AEBSF. Phase contrast images were taken of living cultures on days 312. On day 12 after plating, one set of cultures was incubated with MTT as described below to determine cell viability. A second set of cultures was fixed on day 12 with 70% ethanol and processed for quantitative Alizarin red S staining as described below.
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The abbreviations used are: BMF, biomineralization foci; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride HCl; FBS, fetal bovine serum; BSP, bone sialoprotein; BAG-75, bone acidic glycoprotein-75; BGP, -glycerol phosphate; BSA, bovine serum albumin; CL, total cell layer extract from BGP-treated cultures; CL, total cell layer extract from cultures not treated with BGP; MAA, Maackia amurensis agglutinin; MS/MS, tandem mass spectrometry; LC, liquid chromatography; SKI-1, subtilisin kexin isozyme-1; LCM, laser capture microscope; 1,25D3-MARRS, 1,25-vitamin D3-membrane-associated rapid-response steroid-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MEM, minimal essential medium; MAA, Maackia amurensis agglutinin.
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FIGURE 1. Biomineralization foci can be isolated from total cell layers by laser capture microscopy. A and B, UMR-106-01 osteoblastic cells were cultured in serum-depleted conditions (BSA), or C, the presence of serum (FBS). Cultures were stained with Alizarin red S to detect hydroxyapatite crystals. B, both conditions failed to mineralize in the absence of BGP. Arrows point to mineralized BMF (A and C). Scale bar 500 m. DF, laser capture microscopy of Alizarin red S stained BMF from UMR-106-01 culture. Arrows refer to the same BMF structures in all panels. D, microscopic view of field to be laser-captured. E, appearance of the residual cell layer left behind after laser dissection of mineralized BMF. F, purified BMF temporarily affixed to the cap used for laser capture. Gel images are representative of multiple analyses on two separate BMF preparations. Scale bar 25 m.
FIGURE 2. LCM-captured BMF display distinctive glyco- and phosphoprotein staining patterns compared with the total cell layer fraction. The same amount of protein was applied to each lane. Asterisks emphasize the quantitative enrichment of 75 80-kDa glycophosphoproteins and of a 65-kDa Sypro ruby-stained protein in BMF lanes. Molecular weight estimates refer to blue pre-stained standards co-electrophoresed on the same gel. Results depicted are representative of two separate BMF preparations. Buffer, 0.1 M Tris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide); BMF, represents proteins extracted from purified BMF (see Fig. 1F); CL, cell layer extract of cultures after 24 h mineralization in -glycerol phosphate (see Fig. 1D); CL, cell layer extract of cultures not treated with -glycerol phosphate (see Fig. 1B).
identifications made contain at least two peptides match in the MS/MS scans that meets or exceeds the threshold values for a 95% confidence level. Two-dimensional PAGEGels were run according to the method of Witzmann et al. (32) and stained with either colloidal Coomassie Blue G, Pro-Q Emerald 300 glycoprotein stain (Invitrogen), or Pro-Q Diamond phosphoprotein stain (Invitrogen). PD-Quest (Bio-Rad) software was used to digitally analyze the colloidal Coomassie Blue G-stained gels comparing AEBSFtreated with nontreated cell layer and media fractions to identify proteins differentially expressed in one condition versus another.
RESULTS Mineralization of UMR Osteoblastic Cells Is Unchanged in Serum-depleted ConditionsTo limit contamination by serum proteins in isolated BMF, we tested whether use of serum-free conditions would affect the amount or morphology of mineralization in UMR cultures. No differences were noted in the amount or morphology of mineralized BMF when conditions of serum depletion were compared with serum-replete conditions (compare Fig. 1, A versus C). As expected (22), few mineral crystals are evident when BGP is omitted (Fig. 1B). Quantitation of the amount of Alizarin red stain bound per well also revealed no significant differences (not shown). Manual counts of mineralized BMF formed under serum-containing and serum-depleted conditions showed no statistical difference (103 foci/cm2 6.56 S.D. versus 105 mineralized foci/cm2 6.08 S.D., p 0.486 using one-way analysis of variance followed by Kruskal-Wallis method). These results confirm that the mineralization potential is unchanged in conditions of serum depletion. BAG-75 and BSP and Fragments of Each Are Enriched within Purified Mineralized BMFMineralized BMF, which appear as dark spots about 20 25 m in diameter, were isolated from ethanol-fixed, Alizarin red-stained UMR cultures by laser capture microscopy (Fig. 1, DF). Use of Alizarin red staining for identification provides a direct connection to previous work
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that defined BMF structures as sites of initial mineral crystal nucleation (12, 22). After laser capture of mineralized BMF, Fig. 1E depicts residual holes that are devoid of cells and show the underlying glass surface. Finally, Fig. 1F provides an image of the resultant captured BMF preparation with individual foci arranged in the same relative orientation as they appeared on the original stained slide (Fig. 1D). Visual inspection of captured populations revealed an absence of obvious cellular contamination. Furthermore, attempts at direct isolation of cells from the fixed cell layer using an LCM approach proved unsuccessful demonstrating that the fixed UMR cells adhere too tightly to the glass slide to permit their capture from this surface.3 Following capture, 6200 pooled BMF were extracted by mixing with 0.1 M Tris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide, and then subjected to SDS-PAGE. As controls, UMR culture slides containing the total cell layer along with mineralized BMF (as depicted in Fig. 1D, CL) were processed similarly. Culture slides not treated with -glycerol phosphate and containing nonmineralized pre-BMF represent a second control (as depicted in Fig. 1B, CL). Equal amounts of protein were applied to each lane, and gels were stained as indicated (Fig. 2). SDS-PAGE results show substantial enrichment of 75-kDa glycoproteins and phosphoproteins in the BMF extract when compared directly with the CL control (Fig. 2, see bands with asterisks). Sypro Ruby staining showed enrichment of bands in the 65-kDa range in BMF. In contrast, bands in the 10 15-kDa range appeared to be shared by both the BMF and total cell layer samples (Fig. 2). Although not quantitative, this comparative analysis is designed to identify those proteins substantially enriched within mineralized BMF. Our approach is based upon the hypothesis that BMF are structures assembled for the specific purpose of nucleating hydroxyapatite crystals in culture
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Downloaded from www.jbc.org by guest, on November 13, 2009 FIGURE 3. Comparative immunostaining of LCM-isolated BMF versus total cell layer fractions demonstrates an enrichment of BAG-75 and BSP and their fragments. AE, protein extracts from LCM-captured BMF, cell layer fractions, and a buffer control were electroblotted onto polyvinylidene difluoride membrane and developed with anti-BAG-75 or anti-BSP antibodies. Arrows indicate 4550-kDa fragments of BAG-75 and of BSP enriched in BMF extracts over that in the CL total cell layer. The amount of protein loaded onto gel lanes for blots AC was 6.5 g, for blot D was 13 g, and for blot E was 26 g. Blots D and E were intentionally overdeveloped to detect the BSP fragment. Antibodies used are as follows: anti-BAG-75 protein antibody (number 504); anti-BAG-75 peptide antibody (number 503); and anti-BSP antibody (LF-100). BMF, extract of LCM-captured biomineralization foci; CL, extract of total cell layer from -glycerol phosphate-treated cultures; CL, extract of total cell layer from cultures not treated with -glycerol phosphate; buffer, 0.1 M Tris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide. FH, BAG-75 and BSP are two prominent 75-kDa glycoproteins in rat primary bone extracts. F, rat bone G/E extract was electroblotted and detected either with digoxygenin-labeled MAA lectin or with anti-BAG-75 antibodies (number 504) (immunoblot). The extract was also electrophoresed and stained directly with Stains All dye. G, purified BSP, BAG-75, and mixture of BAG-75 and its 50-kDa fragment were electrophoresed and stained with Stains All. H, purified BSP and a mixture of BAG-75 and its 50-kDa fragment were electroblotted and detected with digoxygenin-labeled MAA lectin. Molecular weight estimates are based on pre-stained standards co-electrophoresed on the same gel.
Target protease(s) Trypsin, chymotrypsin, plasmin, thrombin, kallikrein, proprotein convertases Trypsin, chymotrypsin, and plasmin Papain, trypsin, and plasmin Activated complement protein C1s Cysteine proteases Elastase and elastase-like proteases Matrix metalloproteinases 2, 3, 8, and 9 Furin Thrombin Trypsin-like proteases and some cysteine proteases Plasmin and plasma kallikrein Urokinase plasminogen activator
Range of concentration tested 0.010.4 mM 03 g/ml 100 M 0.1100 g/ml 10 M 100 M 10 M 110 M 0.510 ATUa 100 M 1100 M 1100 M
One antithrombin unit (ATU) will neutralize 1 NIH unit of thrombin at 37 C, based on direct comparison with an NIH thrombin reference standard.
Only one inhibitor, AEBSF, blocked mineral nucleation in BMF (Table 1 and Fig. 4A). AEBSF is a covalent serine protease inhibitor (34) and was capable of completely blocking mineral nucleation at concentrations as low as 0.04 mM. None of the other protease inhibitors tested, which included inhibitors of thrombin, plasmin, plasminogen activator, furin, and matrix metalloproteinases, diminished mineralization in the UMR system when used at their optimal recommended dosage (Table 1). When added at 64 h after plating, AEBSF was similarly effective regardless of whether serum was included in the culture media or not (Fig. 4A), indicating that the source of the mineralization-related, AEBSF-sensitive protease is the UMR 106 cells themselves. However, the time at which AEBSF was added dramatically influenced the outcome. Assuming a control mineral level represented by 150 170 nmol of Alizarin red dye/well, the inhibitor was 10-fold less effective if present during the period in which the cells are actively proliferating and differentiating (44 64 h after plating) rather than during the mineralization period (64 88 h after plating) (22) (Fig. 4A). To exclude the possibility that the effects of AEBSF were because of cell toxicity, AEBSF-treated and nontreated control cultures were analyzed using the MTT assay, a widely accepted assay for cell viability that measures vital mitochondria (27). As shown in Fig. 4A, AEBSF only shows toxic effects at concentrations above 0.4 mM. This outcome is similar whether cells are grown in serum-sufficient or serum-depleted conditions. Previously, we have shown that mineralization of MC3T3 osteoblastic cells and of primary calvarial osteoblasts also occurred at sites enriched in BAG-75 (12), similar to that in the UMR model. We therefore next determined whether AEBSF could also block mineralization in primary calvarial osteoblasts. Because the length of exposure is necessarily longer than with UMR cells because of the longer time course of this mineralization model, we initially added AEBSF starting at day 0, 3, 6, or 9 and continuing until the end of the 12-day culture period (not shown). Media-containing treatments were changed every 3 days. On this basis, the minimal effective treatment window was found to be between days 9 and 12 (Fig. 4B). Specifically, 0.01 0.1 mM AEBSF was able to block mineralization completely in primary calvarial cultures when scored on day 12 (Fig. 4B and Fig. 5). Parallel assays of primary calvarial osteoblast viability showed that AEBSF had no consistent effect on cell viability (Fig. 4B). Instead, cell viability seemed to exhibit a very
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gradual decrease over the concentration range tested and approached 90% of the control value at 0.1 mM AEBSF. Mineralization of primary calvarial osteoblast cultures occurs within multilayered nodules (Fig. 5), whereas in UMR 106 cultures, it is initiated within spherical 20 25-m BMF structures. Despite the clear morphological differences between these two sites of mineral nucleation, AEBSF was similarly effective in both systems (Fig. 4, A and B). Uptake of a 75-kDa Phosphoglycoprotein Band in the Cell Layers of Mineralizing Cultures Is Blocked by AEBSFTo examine the effect of AEBSF on the protein distribution within mineralizing UMR 106 cultures, cells were grown until 64 h at which time they were re-fed using one of the following four different media conditions: 1) 7 mM BGP only (BGP); 2) 7 mM BGP and 0.1 mM AEBSF (BGP AEBSF); 3) the absence of both BGP and AEBSF (BGP), and 4) with 0.1 mM AEBSF present but no BGP (BGP AEBSF). Cell layer extracts and media fractions from all four conditions were then compared using SDS-PAGE followed by staining or immunoblotting. Following mineralization, the cell layer was extracted with either a 50 mM EDTA/CHAPS detergent solution or with an 8 M urea, 0.5% SDS, 50 mM EDTA extraction solution (see Experimental Procedures for details). Solubility was defined by ultracentrifugation at 108,000 g for 1 h. Gel electrophoresis revealed that during the 24-h mineralization period (BGP AEBSF), a 75-kDa glyco- and phosphoprotein band is lost from the media fraction (Fig. 6A, arrows). At the same time, a similarly sized glycoprotein band appears in the cell layer fraction. This suggests uptake by the mineralizing cell layer, presumably into the BMF, because a similar glycophosphoprotein band was shown in Fig. 2 to be specifically enriched in mineralized LCMcaptured BMF. The 75-kDa glycoprotein band is likely composed of BAG-75 and BSP because they are the only two proteins of this molecular weight in total bone extracts shown to react with digoxygenin-labeled MAA lectin (see Fig. 3H). The 75-kDa phosphoprotein band is presumed to be predominantly composed of BAG-75 because BSP from bone exhibits a low phosphate content, whereas BAG-75 contains 44 phosphates/mol (35). Loss from the media fraction only occurs when mineralization is ongoing and not when it is blocked by inclusion of AEBSF or when BGP is omitted (Fig. 6A). Although similar analyses of the cell layer demonstrate that a
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FIGURE 5. Phase contrast microscopy of mineralized and AEBSF-treated primary calvarial osteoblastic cultures. Primary calvarial osteoblastic cells were harvested and cultured as described under Methods. On day 9 after plating, some of the culture wells were treated with 0.01 mM AEBSF, whereas control cultures were re-fed normal media. Unstained cultures were photographed on day 12. A, phase contrast image of control cultures. Mineralized nodules (arrows) appear as dark deposits under these conditions. B, phase contrast image of culture treated with 0.01 mM AEBSF. No mineralized nodules were visible. Results shown are representative of multiple wells and were consistent in three separate experiments. Scale bar 500 m.
FIGURE 4. AEBSF inhibits mineral nucleation both in UMR 106 osteoblastic cultures and in primary mouse calvarial osteoblasts. A, with UMR cells, AEBSF blocks mineralization similarly in both serum-containing and serumdepleted conditions, while displaying higher effectiveness with mineralization competent cultures. The amounts of Alizarin red S bound to calcium phosphate crystals within BMF and cell viability assessed with the MTT assay were plotted versus the concentration of AEBSF added to cultures. AEBSF was present two times during the culture model, e.g. from 44 to 64 h after plating and from 64 to 88 h after plating. A 4-fold increase in sensitivity was observed in converting from serum-sufficient conditions to serum-depleted conditions, whereas a 10-fold increase in effectiveness was obtained when comparing 64 88 h versus 44 64 h cultures. For 64 88 h cultures: , MTT absorbance in serum-depleted conditions; f, MTT absorbance in serum-containing media; E, amount of Alizarin red bound in serum-depleted conditions; F, amount of Alizarin red bound in serum-containing media. For 44 64 h cultures: , amount of Alizarin red bound in serum-containing media. (MTT assay results for 44 64-h cultures were essentially identical to those for 64 88 h cultures and were omitted from the graph to maintain clarity.) For Alizarin red S assays, designated results () were significantly different from controls at p 0.05; for MTT cell viability assays, only cultures treated with 1 mM AEBSF (*) differed significantly from controls at p 0.05. UMR culture studies were carried out in triplicate; results shown are representative of four separate experiments. Error bars represent the means S.D. B, AEBSF completely inhibits mineralization within nodules of primary mouse calvarial cultures. MTT assay results and the amount of Alizarin red S bound to mineral deposits within cultures on day 12 are plotted versus the concentration of AEBSF added to cultures on day 9. , MTT absorbance in serum-containing media; E, amount of Alizarin red bound in serum-containing media. For Alizarin red S assays, individual data points () differed significantly from controls at p 0.05; for MTT cell viability assays, results (#) were significantly different from controls at p 0.05. Primary culture studies were done in quadruplicate; results shown are representative of three separate experiments. Error bars represent the mean S.D.
FIGURE 6. One-step extraction of UMR osteoblastic cell layer is unable to account for the quantitative loss of BAG-75 from the media fraction occurring during mineralization. A, extraction of cell layers with 50 mM EDTA/CHAPS extraction buffer (0.1 M Tris acetate buffer (pH 7.8), containing with 0.5% octyl glucoside, 0.05% SDS, 0.05 M EDTA, and 0.02% sodium azide) reveals the loss of 75-kDa glycophosphoproteins from the media and the subsequent uptake of this band into the cell layer. Treatment of the cultures with AEBSF blocks the cell layer uptake of this same band. A, media and cell layer extracts from cell cultures treated with or without -glycerol phosphate and with or without AEBSF. B, extraction of cell layers with 8 M urea, 50 mM EDTA, and 0.5% SDS reveals BAG-75 is lost from the media of -glycerol phosphate-treated cultures, while unaccounted for in the cell layer extract from the same cultures. Recovery of full-length 75-kDa BSP from the cell layer of -glycerol phosphate treated cultures, along with that for conditioned media, is comparable with that without -glycerol phosphate; however, no BSP fragment (4550-kDa) was detected in the cell layer fraction.
75-kDa glycoprotein is taken up only when mineralization is progressing, a comparable increase in phosphoprotein (e.g. BAG-75) staining is not observed (Fig. 6A, arrows). These conclusions were confirmed when similar one-step extracts were probed with monospecific antibodies (Fig. 6B). Although approximately one-half of the BSP is lost from the
media fraction during mineralization (BGP), a comparable amount of BSP became associated with the cell layer. Although BAG-75 protein was also lost from the media fraction only when mineralization occurred (media, BGP), its recovery in the cell layer fraction was lower than expected. This is contrary to the known presence of BAG-75 antigen in BMF and nodular complexes prior to and during their mineralization in osteoblastic cell cultures (12). As a result, we
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Apparent mass
Da
Expected pI
Method(s) for identification Differential staining after two-dimensional SDS-PAGE and mass spectroscopy Differential staining after two-dimensional SDS-PAGE and mass spectroscopy Differential staining of two-dimensional SDS after PAGE and mass spectroscopy One-dimensional SDS-PAGE immunoblotting One-dimensional SDS-PAGE immunoblotting
Peptides identified (Mascot score) FDVEPDTYCR (59) TGDLDLPSPASGTSLK (49) SGTLQSNFCSSSLVVTGTVK (75) LNFAVASR (63) YGVSGYPTLK (65) LAPEYEAAATR (88) FAHTNVESLVK (74) DLFSDGHSEFLK (72) DIELVMSQANVSR (75) SPASDTYIVFGEAK (79) NILFVITKPDVYK (80) NAa NA
48,000 50,000
8.5 5.4
Nascent polypeptide associated complex, chain Bone acidic glycoprotein-75 Bone sialoprotein
itor, was able to specifically block mineral nucleation within BMF in UMR 106 cultures and in mineralization nodules in primary calvarial osteoblastic cultures. In the UMR model, the inhibitor was 10-fold more effective if present during the mineralization phase (64 88 h after plating) rather than during the proliferation/differentiation phase (44 64 h after plating). Similarly, in primary osteoblastic cultures, AEBSF appeared to block a step just prior to mineral crystal nucleation. Fourth, when mineralization was blocked by AEBSF, cleavage of BAG-75 and BSP was also inhibited. Furthermore, two-dimensional SDS-PAGE comparisons of UMR culture fractions in the presence and absence of AEBSF identified three other proteins (procollagen C proteinase enhancer protein, membrane-associated rapid-response steroid receptor, and nascent polypeptide associated complex chain) whose cleavage was also blocked by this inhibitor. Fifth, although BSP and BAG-75 become enriched within BMF during their mineralization, they originally are also present in the media compartment. Taken together, our results indicate for the first time that cleavage of BAG-75, BSP, procollagen C proteinase enhancer protein, 1,25vitamin D3-membrane-associated rapid-response steroidbinding protein, and nascent polypeptide associated complex chain and mineral nucleation within biomineralization foci are associated with an AEBSF-sensitive protease produced by osteoblastic cells. The data suggest these proteolytic events may be a permissive step for mineralization to proceed. Selective proteolysis or fragmentation plays a critical role in many biological processes, e.g. blood coagulation, fertilization, and complement activation, where it represents a means to regulate protein function through activation or inactivation. Serine proteases play a key role in tumor-induced bone formation and tooth mineralization. Enamel mineralization requires serine proteases enamelysin (MMP-20) and kallikrein 4 (38). Enamelysin catalyzes the cleavage of amelogenin, one of the main structural proteins forming enamel. Kallikrein 4 is responsible for degradation of enamel proteins, which results in their removal from the matrix allowing the enamel layer to fully mineralize. Previously, we showed stromelysin cleaved BAG-75 into a 50-kDa fragment in vitro (39); a similar 50-kDa fragment is found in calcified tissue and in serum (24). Preliminary data show that the level of 50-kDa fragment in serum correlates
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directly with bone formation following ovariectomy of rats (40). BSP has also been shown to undergo proteolysis by an endogenous protease in UMR 106 cells yielding a fragment of 47 kDa (41). This fragment is similar in size to that produced here by the AEBSF-sensitive protease. Although cleavage of these phosphoproteins has been noted previously (above), our findings are the first to link BSP and BAG-75 fragments specifically to the site of mineral nucleation. Because the identity of the protease responsible for endogenous fragmentation of BSP and BAG-75 in bone is presently unknown, we surveyed a wide range of competitive and noncompetitive inhibitors against serine and cysteine proteases and matrix metalloproteinases. Of all the inhibitors tested, AEBSF was the only one that had a specific effect on mineralization. Some like Pefabloc urokinase-type plasminogen activator were found to block mineralization nonspecifically because of their associated toxicity. This was identified by separate, parallel assays of cell viability. For AEBSF, toxicity was observed only at concentrations 100-fold above those found to completely block mineral nucleation in BMF, e.g. 0.01 mM. In addition to full-length forms, EDTA extracts of the cell layer contained a 50-kDa fragment of BAG-75 and a 45-kDa fragment of BSP under mineralizing conditions only. A major source of BAG-75 and BSP was the conditioned media, because in the absence of BGP, full-length proteins were localized predominantly to the media. We have previously shown that addition of a phosphate source to 64-h cultures (mineralizationcompetent) is necessary to induce BSP uptake by BMF and their subsequent mineralization (12, 22); in the absence of phosphate, BAG-75 enriched BMF remain unmineralized. The 10-fold greater effectiveness of AEBSF when added to 64-h versus 44-h UMR cultures suggests that the inhibitor acts late in the differentiation phase just prior to mineral nucleation. Similarly, in primary calvarial cultures, late addition of AEBSF on day 9 proved just as effective as on days 3 or 6 during the differentiation phase (5, 25, 26, 42) suggesting that AEBSF acts preferentially during the period immediately before mineral crystal nucleation in nodules. In this way, both the UMR 106 and primary osteoblast culture models exhibited similar sensitivities to AEBSF.
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